The sequencing of individual DNA strands with nanopores is under investigation as a rapid, low-cost platform in which bases are identified in order as the DNA strand is transported through a pore under an electrical potential. Although the preparation of solid-state nanopores is improving, biological nanopores, such as ␣-hemolysin (␣HL), are advantageous because they can be precisely manipulated by genetic modification. Here, we show that the transmembrane ␤-barrel of an engineered ␣HL pore contains 3 recognition sites that can be used to identify all 4 DNA bases in an immobilized single-stranded DNA molecule, whether they are located in an otherwise homopolymeric DNA strand or in a heteropolymeric strand. The additional steps required to enable nanopore DNA sequencing are outlined.␣-hemolysin ͉ DNA sequencing ͉ genomics ͉ protein engineering ͉ protein pore

Droplet interface bilayers (DIBs) provide a superior platform for the biophysical analysis of membrane proteins. The versatile DIBs can also form networks, with features that include built-in batteries and sensors.

Despite recent improvements in sequencing methods, there remains a need for assays that provide high sequencing depth and comprehensive variant detection. Current methods 1 - 4 are limited by the loss of native modifications, short read length, high input requirements, low yield, or long protocols. Here, we describe nanopore Cas9-targeted sequencing (nCATS), an enrichment strategy that uses targeted cleavage of chromosomal DNA with Cas9 to ligate adaptors for nanopore sequencing. We show that nCATS can simultaneously assess haplotype-resolved single-nucleotide variants (SNVs), structural variations (SVs) and CpG methylation. We apply nCATS to four cell lines, a cell-line-derived xenograft, and normal and paired tumor/normal primary human breast tissue. Median sequencing coverage was 675X using a minION flow cell and 34X using the smaller flongle flow cell. nCATS requires only ~3μg of genomic DNA and can target a large number of loci in a single reaction. The method will facilitate the use of long-read sequencing in research and in the clinic.

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Recently, we demonstrated that submicrolitre aqueous droplets submerged in an apolar liquid containing lipid can be tightly connected by means of lipid bilayers [1][2][3][4][5] to form networks [4][5][6] . Droplet interface bilayers have been used for rapid screening of membrane proteins 7,8 and to form asymmetric bilayers with which to examine the fundamental properties of channels and pores 9 . Networks, meanwhile, have been used to form microscale batteries and to detect light 4 . Here, we develop an engineered protein pore with diode-like properties that can be incorporated into droplet interface bilayers in droplet networks to form devices with electrical properties including those of a current limiter, a half-wave rectifier and a full-wave rectifier. The droplet approach, which uses unsophisticated components (oil, lipid, salt water and a simple pore), can therefore be used to create multidroplet networks with collective properties that cannot be produced by droplet pairs.To obtain directional ionic current flows in droplet networks ( Fig. 1), we constructed a diode-like pore from staphylococcal a-haemolysin (aHL). aHL forms a heptameric protein pore 10 that inserts vectorially into lipid bilayers 11 . The crystal structure of the pore reveals a 14-stranded transmembrane b barrel capped by an extramembraneous domain, which contains a roughly spherical cavity 10 ( Fig. 2a, left). The wild-type (WT) pore is a 'blank slate' for protein engineering with properties similar to those of an electrolyte-filled tube; it is weakly rectifying and weakly anion selective and gates only at extreme applied potentials of either polarity 12 .aHL has been modified by mutagenesis or targeted chemical modification to form pores with a wide range of properties [13][14][15][16] , but none has exhibited sufficient rectification for our purpose. We had, however, noticed that aHL pores with positively charged side chains projecting into the lumen of the transmembrane b barrel tended to gate (open and close) at negative potentials. Therefore, in an attempt to obtain a fully rectifying pore, we tested an extreme version of aHL in which seven residues were replaced with arginines (7R-aHL) to yield a heptameric pore in which 49 additional positively charged side chains were located within the barrel (Fig. 2a, right). In 1 M KCl, 25 mM Tris HCl at pH 8.0, 100 mM, in planar lipid bilayers, the 7R-aHL pore has a unitary conductance of 0.95 + 0.01 nS (þ50 mV, n ¼ 8). The conductance of the WT pore under the same conditions is similar (0.99 + 0.02 nS, n ¼ 4), which suggests, surprisingly, that the drastically altered 7R-aHL pore is properly formed. The current-voltage (I-V) characteristics of 7R-aHL in 1 M KCl, however, showed virtually complete current rectification (Fig. 2b,c). At positive applied potentials, 7R-aHL remained in an open form with a stable steady-state current and infrequent short-lived closures of less than 10 ms. By contrast, at negative applied potentials, the pore was closed, with occasional brief current spikes ascriba...

Aqueous droplets in oil that are coated with lipid monolayers and joined through interface bilayers1,2 are useful for biophysical measurements on membrane proteins2–5. Further, functional networks of droplets that can act as light sensors, batteries and electrical components can be made by incorporating pumps, channels and pores into the bilayers2,6. These networks of droplets mimic simple tissues7, but so far have not been used in physiological environments because they have been constrained to a bulk oil phase. Here we form multisomes: networks of aqueous droplets with defined compositions within small drops of oil in water (Fig. 1a). The encapsulated droplets can communicate with each other and with the surrounding aqueous environment through membrane pores. The contents in the droplets can be released by changing the pH or temperature of the surrounding solution. Multisomes constitute a multi-compartment protocellular chassis7–9 with potential medical applications.

Ribonucleic acid sequencing can allow us to monitor the RNAs present in a sample. This enables us to detect the presence and nucleotide sequence of viruses, or to build a picture of how active transcriptional processes are changing – information that is useful for understanding the status and function of a sample. Oxford Nanopore Technologies’ sequencing technology is capable of electronically analysing a sample’s DNA directly, and in real-time. In this manuscript we demonstrate the ability of an array of nanopores to sequence RNA directly, and we apply it to a range of biological situations. Nanopore technology is the only available sequencing technology that can sequence RNA directly, rather than depending on reverse transcription and PCR. There are several potential advantages of this approach over other RNA-seq strategies, including the absence of amplification and reverse transcription biases, the ability to detect nucleotide analogues and the ability to generate full-length, strand-specific RNA sequences. Direct RNA sequencing is a completely new way of analysing the sequence of RNA samples and it will improve the ease and speed of RNA analysis, while yielding richer biological information.

In this paper, we give an overview of our studies by static and time-resolved X-ray diffraction of inverse cubic phases and phase transitions in lipids. In [section sign] 1, we briefly discuss the lyotropic phase behaviour of lipids, focusing attention on non-lamellar structures, and their geometric/topological relationship to fusion processes in lipid membranes. Possible pathways for transitions between different cubic phases are also outlined. In [section sign] 2, we discuss the effects of hydrostatic pressure on lipid membranes and lipid phase transitions, and describe how the parameters required to predict the pressure dependence of lipid phase transition temperatures can be conveniently measured. We review some earlier results of inverse bicontinuous cubic phases from our laboratory, showing effects such as pressure-induced formation and swelling. In [section sign] 3, we describe the technique of pressure-jump synchrotron X-ray diffraction. We present results that have been obtained from the lipid system 1:2 dilauroylphosphatidylcholine/lauric acid for cubic-inverse hexagonal, cubic-cubic and lamellar-cubic transitions. The rate of transition was found to increase with the amplitude of the pressure-jump and with increasing temperature. Evidence for intermediate structures occurring transiently during the transitions was also obtained. In [section sign] 4, we describe an IDL-based 'AXcess' software package being developed in our laboratory to permit batch processing and analysis of the large X-ray datasets produced by pressure-jump synchrotron experiments. In [section sign] 5, we present some recent results on the fluid lamellar-Pn3m cubic phase transition of the single-chain lipid 1-monoelaidin, which we have studied both by pressure-jump and temperature-jump X-ray diffraction. Finally, in [section sign] 6, we give a few indicators of future directions of this research. We anticipate that the most useful technical advance will be the development of pressure-jump apparatus on the microsecond time-scale, which will involve the use of a stack of piezoelectric pressure actuators. The pressure-jump technique is not restricted to lipid phase transitions, but can be used to study a wide range of soft matter transitions, ranging from protein unfolding and DNA unwinding and transitions, to phase transitions in thermotropic liquid crystals, surfactants and block copolymers.